In the treatment of tumors, autoimmune diseases, allergies and tissue rejection reactions, it is a disadvantage that the currently available medicaments, such as chemotherapeutic agents, corticosteroids and immunosuppressive agents, have a potential of side effects which is sometimes considerable, due to their relative non-specificity. It has been attempted to moderate this by various therapeutical concepts. Especially the use of immunotherapeutic agents is an approach, which resulted in an increase of the specificity of medicaments, especially in tumor treatment.
Immunotoxins are proteins used to treat e.g. cancer that are composed of an antibody or a fragment thereof linked to a toxin. The immunotoxin binds to a surface antigen on a cancer cell, enters the cell, and kills it. The most potent immunotoxins are made from bacterial and plant toxins. Refinements over many years have produced recombinant immunotoxins; these therapeutic proteins are made using protein engineering. Individual immunotoxins are designed to treat specific cancers. To date, most success has been achieved treating hematologic tumors. Obstacles to successful treatment of solid tumors include poor penetration into tumor masses and the immune response to the toxin component of the immunotoxin, which limits the number of doses that can be given. Strategies to overcome these limitations are being pursued.
The protein-based cell toxins which have been mostly used to date and are thus best characterized, are the bacterial toxins diphtheria toxin (DT) (Beaumelle, B. et al. 1992; Chaudhary, V. et al. 1990; Kuzel, T. M. et al. 1993; LeMaistre, C. et al. 1998), Pseudomonas exotoxin A (PE) (Fitz Gerald, D. J. et al. 1988; Pai, L. H. and Pastan, I. 1998), and the plant-derived ricin-A (Engert, A. et al. 1997; Matthey, B. et al. 2000; O'Hare, M. et al. 1990; Schnell, R. et al. 2000; Thorpe, P. E. et al. 1988; Youle, R. J. and Neville, D. M. J. 1980). The mechanism of cytotoxic activity is the same in all of these toxins despite of their different evolutionary backgrounds. The catalytic domain inhibits protein biosynthesis by a modification of the elongation factor EF-2, which is important to translation, or of the ribosomes directly, so that EF-2 can no longer bind (Endo, Y. et al. 1987; Iglewski, B. H. and Kabat, D. 1975).
In most of the constructs employed to date, the systemic application of immunotoxins results in more or less strong side effects. In addition to the “vascular leak” syndrome (Baluna, R. and Vitetta, E. S. 1997; Schnell, R. et al. 1998; Vitetta, E. S. 2000), thrombocytopenia, hemolysis, renal insufficiency and sickness occur, depending on the construct employed and the applied dosage. Dose-dependent and reversible liver damage could also be observed (Battelli, M. G. et al. 1996; Grossbard, M. L. et al. 1993; Harkonen, S. et al. 1987). In addition to the documented side effects, the immunogenicity of the constructs employed to be observed in the use of the immunoconjugates or immunotoxins is the key problem of immunotherapy (Khazaeli, M. B. et al. 1994). This applies, in particular, to the humoral defense against the catalytic domains employed, such as ricin (HARA) (Grossbard, M. L. et al. 1998), PE (Kreitman, R. J. et al. 2000), or DT (LeMaistre, E. F. et al. 1992). Theoretically, all non-human structures can provoke an immune response. Thus, the repeated administration of immunotoxins and immunoconjugates is subject to limitations. A logical consequence of these problems is the development of human immunotoxins, now named human cytolytic fusion proteins (Rybak, S. et al. 1992).
For example, cytotoxic lymphocyte granules contain perforin, a pore-forming protein, and a family of serine proteases, termed granzymes. Granzymes are serine proteases that are mainly expressed in cytotoxic T lymphocytes and natural killer cells.
Granzymes are involved in cell death by apoptosis (Coughlin et al, 2000) but also have other functions, including the regulation of B-cell proliferation, cleavage of extracellular matrix proteins, induction of cytokine secretion, activation of cytokines, control of viral infections (Trapani, 2001). For example, the serine protease granzyme B (GrB) (Lobe et al., 1986; Schmid and Weissman, 1987; Trapani et al., 1988) is integrally involved in apoptotic cell death induced in target cells upon their exposure to the contents of lysosome-like cytoplasmic granules (or cytolytic granules) found in cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells (Henkart, 1985; Young and Cohn, 1986; Smyth and Trapani, 1995).
WO 01/80880 A1 discloses the use of the serine protease granzyme B in a cytolytic fusion protein (immunoprotease). The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. However, granzyme B has a number of efficient natural inhibitors like Serpin B9 that prevent granzyme B-mediated apoptosis in certain cell types.
Thus, the technical problem underlying the present invention was to provide novel and improved cytolytic fusion proteins for the treatment of a disease.